This is an invention the field of amplification of DNA by polymerase chain reaction (PCR).
PCR is a method that typically utilizes two oligonucleotide primers to amplify a DNA segment &gt;1 million-fold. The two primers anneal to opposing strands of DNA at positions which span a target sequence of interest. A DNA polymerase is used for sequential rounds of template dependent synthesis of the DNA sequence. The PCR method is more fully described in U.S. Pat. No. 4,683,202, issued Jul. 28, 1987, the disclosure of which is incorporated herein.
PCR can be adapted for the rapid detection of single-base changes in genomic DNA by using specifically designed oligonucleotides in a method called PCR amplification of specific alleles (PASA) (Sommer et al. 1989; Sarkar et al. 1990; Wallace et al. U.S. Pat. No. 5,639,611, issued Jun. 17, 1997). This rapid method is also known as allele-specific amplification (ASA), allele-specific PCR, and amplification refractory mutation system (ARMS) (Newton et al. 1989; Nichols et al. 1989; Wu et al. 1989). For this technique an oligonucleotide primer is designed to match one allele perfectly but mismatch the other allele at or near the 3' end, thereby preferentially amplifying one allele over the other. PASA assays can be developed for assaying virtually all alleles (Sommer et al. 1992). However, each PASA reaction provides information on the presence or absence of only one allele. Two PASA reactions must be performed to determine the zygosity of any sequence change.
To detect zygosity in one PCR reaction, PCR amplification of multiple specific alleles (PAMSA) utilizes three primers in one reaction to generate two allele-specific segments that differ sufficiently in size to be distinguished by agarose gel electrophoresis (Dutton and Sommer 1991). However, problems arise from PAMSA because of differences in length, and hence, amplification efficiency of the allele-specific primers. A similar approach-termed competitive oligonucleotide primary (COP)-utilized primers that mismatched the undesired allele within the middle rather than the end of the oligonucleotide (Gibbs et al. 1989; Ruano and Kidd 1989).
Tetra-primer PCR is a method by which two allele-specific amplifications occur in opposite directions (Ye et al. 1992). Tetra-primer PCR and the method of this invention, which we have named Bi-Directional Polymerase Chain Reaction Amplification of Specific Alleles (Bi-PASA), both rely on allele-specific PCR to amplify two alleles simultaneously and in opposite directions. However, the methods differ in the following ways. (1) In tetra-primer PCR, the allele specificity of the inner primers derive from mismatches in the middle of two complementary primers, whereas, in Bi-PASA, the mismatches are at (or near) the 3' end of the primers. (2) The inner primers in Bi-PASA have short complementary segments and G+C-rich tails to efficiently switch from template-based amplification to self-amplification and to prevent megapriming. (3) Tetra-primer PCR utilizes two annealing conditions of high and low stringency, whereas Bi-PASA utilizes a constant annealing temperature. (4) The inner primers used in tetra-primer PCR are concentrated 35-fold more than the outer primers, whereas in Bi-PASA the primers are of similar concentration.